The field of reversible protein acetylation has rapidly expanded in the last 4 years, giving rise to new fundamental questions and further highlighting recalcitrant ones for this evolutionarily conserved post- translational modification. From acetyl-proteomic studies, it has become evident that lysine acetylation is widespread occurring throughout the cell on hundreds of proteins. In this competitive renewal proposal, we will address these salient questions by employing global analysis of acetylation by defining regulatory mechanisms and by investigating specific examples of functional consequences of site-specific modification. The NAD+- dependent deacetylases (sirtuins) have emerged as important regulators of major biological processes, including genome maintenance, transcription, and metabolism. However, the regulation of sirtuin activity remains poorly defined. Recently, we have discovered that Sirtuin-6 (SIRT6) is activated directly by certain fatty acids. Here, we will provide the mechanistic basis and biological consequence of this activation, as well as explore this regulatory mode among all mammalian sirtuins, particularly those that display general deacylase activity (Aim 1). The lack of direct evidence for acetyltransferes in the mitochondrial has led to the possibility that substantial protein acetylation is non-enzymatic in this organelle. In this proposal, we will determine the chemical reactivities and acetylation kinetics of native mitochondrial proteins and explore the possibility that increased acetyl-CoA flux/levels are responsible for increase acetylation without the requirement for acetyltransferases (Aim 2). We have recently mapped in multiple tissues mitochondrial acetylation sites that are regulated by mouse SIRT3. The results revealed that SIRT3 controls protein acetylation in core metabolic pathways as well as tissue specific enzymes. How these acetylations control metabolism is unknown. Here, we will determine the molecular functions of reversible acetylation on mitochondrial proteins/enzymes that serve as hubs of metabolism and mitochondrial function (Aim 3). Taken together, this proposal will define fundamental principles that drive acetylation, determine regulatory mechanisms that control deacetylase activity, and reveal the molecular basis of altered activity by site-specific acetylation.